title: Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase.
abstract: Trypanosoma cruzi, the agent of Chagas disease, expresses on its surface a trans-sialidase that catalyzes preferentially the transference of alpha-2,3-linked sialic acid to acceptors containing terminal beta-galactosyl residues, instead of the typical hydrolysis reaction, found in most sialidases. The trans-sialidase is responsible for the acquisition of the host sialic acid by this protozoan parasite, which does not synthesize sialic acids. Here, we have studied some kinetic properties of a recombinant trans-sialidase expressed in Escherichia coli. We found that it has sequential-type kinetics for the transferase reaction, as shown for the parasite-derived enzyme. The rates of sialic acid transfer to water (hydrolysis), and to beta-galactosyl residues have a unique behavior with respect to the reaction temperature. While the hydrolysis rate of sialyllactose increases continuously up to 35 degrees C, the temperature for the maximal rate of trans-glycosylation depends on the acceptor concentration. At low acceptor concentrations the rate of trans-glycosylation is maximal at 13 degrees C and independent of the amount of sialic acid donors. With increasing acceptor concentrations, maximal rates of trans-glycosylation are shifted to higher temperatures. This finding is explained by an 8-fold increase in the Km for the acceptor from 13 degrees C to 33 degrees C. Differences in hydrolysis and transfer rates were also obtained by using 4-methylumbelliferyl-N-acetyl-neuraminic acid. However, its hydrolysis rate is much higher than the rate of transference to lactose, suggesting that a long-lived enzyme-sialosyl intermediate is not formed. In addition, lactose does not increase the rate of methyl-umbelliferone release at any temperature, indicating that the rate limiting step is the aglycon release. Based on these results we propose that transglycosylation in T. cruzi sialidase is favored by the existence of a binding site for beta-galactosyl residues, which accepts the new glycosidic bond as sialic acid is released from the donor. With increasing temperature the affinity for the acceptor decreases, resulting in a concomitant increase in the rate of transfer to water, which, in turn, can be suppressed by increasing the acceptor concentration.
authors.affiliation: Departamento de Microbiologia, Immunologia e Parasitologia, Escola Paulista de Medicina, UNIFESP, São Paulo, Brazil.
authors.forename: M
authors.initials: M
authors.lastname: Ribeirão
authors.forename: V L
authors.initials: VL
authors.lastname: Pereira-Chioccola
authors.forename: D
authors.initials: D
authors.lastname: Eichinger
authors.forename: M M
authors.initials: MM
authors.lastname: Rodrigues
authors.forename: S
authors.initials: S
authors.lastname: Schenkman
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli; D005456:Fluorescent Dyes; D005690:Galactose / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007785:Lactose / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009439:Neuraminidase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014349:Trypanosoma cruzi / Q000201:enzymology*
chemical_list: D005456:Fluorescent Dyes; D006023:Glycoproteins; D011994:Recombinant Proteins; D006923:Hymecromone; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; C069715:trans-sialidase; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid; D007785:Lactose; D005690:Galactose